(1) Field of the Invention
The present invention relates to an immunoassay method and test kit for detecting antibodies to at least one protein of Sarcocystis neurona produced in an equine suspected of harboring the Sarcocystis neurona which causes myleoencephalitis in the equine. The disease is debilitating and may frequently be fatal. The preferred immunoassay method uses preblocking of a Western blot membrane containing at least one known identifying antigen of Sarcocystis neurona with unlabeled antibodies of a Sarcocystis sp. other than Sarcocystis neurona in order to prevent false positive, non-specific binding of a labeled antibody to the protein (s).
(2) Description of the Related Art
A mammalian body relates to the presence of foreign antigens by producing antibody molecules from its lymphocyte cells. Antibodies have the property of selectively binding to certain distinctive sites, known as determinants on antigens, thereby rendering the antigens innocuous. The antibodies have a physical affinity for specific determinants or epitopes of antigenic material. A reaction between an antibody and a determinant on an antigen for which the antibody is specific results in an adduct, commonly referred to as an "immunocomplex". The formation of such complexes makes possible a wide variety of assays for antigenic material. Such assays are known generically as immunoassays.
Immunoassays have replaced other procedures used for in vitro diagnostic methods to detect or quantitate a variety of antigens and/or antibodies in fluids and, particularly, body fluids such as blood serum, urine or spinal fluid with important biologic or pharmacologic properties. The high levels of sensitivity and specificity achieved with immunoassays result from the specific , high-affinity, reversible binding of antibodies and antigens, and from the existence of methods for attachment of sensitive detectable labels (radioactive isotopes, fluorophores, ferritin, free radicals, bacteriophages and enzymes) to antibodies or antigens. Enzymes are most commonly used today.
Immunoassay techniques are based upon the complex binding of the antigenic substance being assayed (analyte) with an antibody or antibodies in which one or the other member of the complex may be labeled, permitting the detection and/or quantitative analysis of the target substance by virtue of the label activity associated with the labeled antigen complex or antibody. Immunoassays are generally classified into two groups: the heterogeneous immunoassay in which a labeled antigen or antibody is separated from the labeled antigen-antibody complex before measurement of label activity in either fraction; and the homogeneous immunoassay in which the activity of labeled antigen is measured in the presence of labeled antigen-antibody complex.
Two such diagnostic assay techniques used to determine the presence or amount of antigen in body fluids are generally known as "competitive" assays and "non-competitive" or "sandwich" assays. Typically, in "competitive" assay techniques, an unlabeled antibody or antigen preparation bound to a solid support or carrier is first reacted with a labeled antigen or antibody reagent solution and then with the body fluid sample wherein the antigen or antibody in the sample competes with the labeled antigen for sites on the supported antibody or antigen. The amount of labeled antigen reagent displaced indicates the quantity of antigen present in the fluid sample to be detected.
In the case of a "sandwich" or "non-competitive" assay, a quantity of unlabeled polyclonal or monoclonal antibody or antigen bound to a solid-support or carrier surface, is reacted with a body fluid sample being evaluated for antigens or antibodies, and then, after suitable incubation time and washing, the sample is further incubated with a solution of labeled anti-antibody. The labeled antibody bound to the solid phase in an antibody-antigen-antibody sandwich or the amount of unbound labeled antibody or antigen in the liquid phase would be determined as a measure of the presence of antigen or antibody in the test sample.
Thus the analyte can be an antibody or an antigen which has produced antibodies in the host.
The problem in the prior art is that some of the antibodies in samples recognize non-unique epitopes of proteins in the immunoassay and, thus, are non-specific. This can render the immunoassay non-specific. In the prior art this problem is referred to as "cross-reactivity". The result of cross-reactivity is that the immunoassay fails to accurately measure the specific analyte to be detected.
Various techniques have been used to prevent non-specific reactions with the analyte. A polyclonal antibody (derived by exposing an animal to the antigen and then separation of the antibodies from the blood serum) can be reacted with reagents which bind non-specific proteins for removal. Monoclonal antibodies (from a fusion of myeloma cells and cells of the animal which produce the monoclonal antibody) can be used to produce more specific binding. The monoclonal antibodies still do not necessarily block non-specific reactions where the antibody recognizes a common protein epitope of the biological sample to be tested. Various blocking agents containing proteins, such as milk or gelatin, are used to block non-specific sites which might cause the antibody to bind non-specifically. These help, but do not solve the problem.
The etiologic agent of equine protozoal myeloecephalitis (EPM) has been shown to be Sarcocystis neurona, (Dubey, J. P., et al., J. Parasitol. 77:212-218 (1991)). Similar neurological disease has been described in one horse due to Neospora spp. (Marsh, A. E., et al., J. Am. Vet. Med. Assoc. 190:1907-1913 (1996)). S. neurona utilizes the opossum (Didelphis virginiana) as its definitive host (Fenger, C. K., et. al., J. of Parasitology 81:916-919 (1995)); Fenger, C. K., et al., Vet. Parasitol. 68:199-213 (1997); Conference of Research workers in Animal Diseases-Abstract 162, November 1997). The opossum passes infective sporocysts into the environment in its feces. Horses and ponies can become infected by ingesting sporocysts of S. neurona (Dubey, J. P, et al., J. Parasitol 77:212-218 (1991)), but they are dead end hosts. When the parasite enters the central nervous system of the horse, clinical neurological disease can result. No EPM cases have been reported in horses that have not originated from the Western Hemisphere. This is believed to be due to definitive host specificity of S. neurona, and therefore EPM does not typically occur outside the range of the opossum.
A Western blot test was developed to detect antibodies to S. neurona-specific antigens (approximately 11, 13 and 10.5 kilodaltons) (Granstrom, D. E., et al., J. Vet Diag Invest 5:88-90 (1993)) in cerebrospinal fluid of horses suspected of having EPM. In 1997, a different criteria for a positive test (with reactivity to proteins of approximately 13, 11, 10.5 and 10 kDa) was used by the same laboratory in the only published experimental infection study of Sarcocystis from opossums in horses (Fenger, C. K., et al., Vet Parasitol 68:199-213 (1997)). The Western blot test has also been used to estimate seroprevalence of antibodies to S. neurona in Pennsylvania, Ohio and Oregon (Bentz, B. G., et al., J. Am Vet Med Assoc 210:517-518 (1997); Saville, W. J., et al., JAVMA 210:519-518 (1997); Blythe, L. L., et al., JAVMA 210:525-527 (1997)). Seroprevalence estimates ranged from 22%-65% for various geographic regions sampled, suggesting high rates of infection with S. neurona. These Western blot assays have not been found to be reliable in predicting the presence of Sarcocystis neurona due to cross-reacting antibodies to other Sarcocystis sp. in the equine.
Recent work has indicated that immunodominant proteins of approximately 12 and 29 kDa are specific to S. neurona (Marsh, A. E., et al, J. Am Vet Med Assoc 190:1907-1913 (1996)), which would suggest a different criteria for a positive test result than that which has been used for diagnostic purposes and seroprevalence estimates. It was found that antibody cross-reactivity to other apicomplexian species can occur at or near these bands and therefore may cause false-positive test results.